Aberrant protein-protein interactions are implicated in a number of human diseases, and understanding protein interaction networks has become the subject of intense research in diverse disciplines. Yet, global detection of protein interactions within biological systems poses a significant challenge for current technology. For instance, mass spectrometry combined with tandem affinity purification is a useful tool for investigating protein interactions on a global scale, but this technique fails to recover transient/weak protein interactions-these interactions are lost during the tandem affinity purification steps. In this proposal, we propose to overcome this problem by introducing a chemical cross-linking strategy aimed at preserving transient protein complexes in vivo. Using this strategy, we will attempt to understand the molecular basis for BRCA1, a critical tumor suppressor in the etiology of breast cancer. BRCA1 encodes a ubiquitin ligase, whose enzymatic activity is abolished by mutations found in breast cancer patients. Elucidation of the cellular function of BRCA1 will greatly enhance our knowledge on breast cancer, but our understanding on BRCA1 function is still incomplete. It is necessary to identify the substrates ubiquitinated by BRCA1 and cellular consequences of their ubiquitin modification in order to define the cellular function of BRCA1. Previous attempts to address this issue by conventional mass spectrometry have produced mixed results probably due to the transient nature of the protein interactions involving BRCA1. -Specific Aims- 1. In Vivo Cross-linking &Mass spectrometry in Mammalian Cells An in vivo cross-linking technique that has been developed for the mass spectrometric analyses of the yeast proteome will be optimized for the mammalian cells. 2. Mechanism of the Tumor Suppressor Function of BRCA1 The cellular substrates and interacting proteins for BRCA1 will be determined using a chemical cross-linking strategy. -Relevance- BRCA1 mutations confer 60-85% lifetime risk of developing breast cancer, which is the most common cancer among women in the U.S. BRCA1 is also frequently down-regulated in sporadic cases, indicating its central role in suppressing the cancer. Thus, our proposal aimed at comprehending how BRCA1 inhibits breast cancer is relevant to public health and NIH mission.